DESCRIPTION: (Verbatim from the Applicant's Abstract) An important mechanism of ischemic brain damage is production of reactive O2 species, including superoxide (O2-). Natural defenses against O2- include 3 isozymes of superoxide dismutase (SOD). CuZnSOD and MnSOD have been shown to be important in scavenging O2- produced in the intracellular space. The remaining isozyme, extracellular SOD (EC-SOD), is found only in the extracellular (EC) space. Transgenic (EC-SOD overexpressing) and knockout (EC-SOD deficient) mice and the metalloporphyrin compound Mn-TM-2-PyP (and EC-SOD mimetic) provide novel opportunity to isolate and examine effects of O2- produced in the EC space. We have shown that EC-SOD overexpression reduces both global and focal ischemic injury while EC-SOD deficiency increases focal ischemic injury. The goal of this research is to define mechanisms by which EC-SOD improves ischemic outcome and determine if these properties can be emulated by use of EC-SOD mimetic compounds. Our fundamental postulate is that EC-SOD provides a beneficial effect on ischemic brain by scavenging O2- in the EC space which has been generated by either activated neutrophils/microglia or membrane bound oxidases. We will examine whether reperfusion is required for EC-SOD to affect histologic/behavioral outcome. We will then examine whether EC-SOD deficiency worsens global ischemic injury and whether this deficiency can be corrected by administration of Mn-TM-2-PyP. O2- sensitive microelectrodes, OH microdialysis, and nitrotyrosine assays will be used to determine if manipulation of EC-SOD expression and use of Mn-TM-2-PyP alters O2- concentrations in ischemic brain. The source of extracellular O2- will be examined by comparing histologic/behavioral ischemic outcome in neutrophil depleted EC-SOD transgenic/knockout mice and whether EC-SOD pharmacologic mimetics have efficacy in NADPH oxidase knock-out mice lacking respiratory burst activity. Long-term recovery studies will be performed to assure that neuroprotective effects of EC-SOD are permanent. Immunoblotting techniques will be used to determine if EC-SOD expression is upregulated in post-ischemic brain and whether expressed EC-SOD is intact of cleaved of its heparin binding domain which might facilitate diffusion to sites of inflammation. We believe this work will provide important mechanistic insight into how ischemic outcome is affected by O2- formed in the extracellular space and that this work will provide novel routes of investigation for therapy of ischemic brain injury.